scholarly journals In Vivo DNA Protection by Relaxed-Specificity SinI DNA Methyltransferase Variants

2008 ◽  
Vol 190 (24) ◽  
pp. 8003-8008 ◽  
Author(s):  
Edit Tímár ◽  
Pál Venetianer ◽  
Antal Kiss
Keyword(s):  
Restriction Endonuclease ◽  
Dna Methyltransferase ◽  
Term Survival ◽  
Modification System ◽  
Restriction Modification System ◽  
Low Concentrations ◽  
Compatible Plasmid ◽  
Restriction Modification

ABSTRACT The SinI DNA methyltransferase, a component of the SinI restriction-modification system, recognizes the sequence GG(A/T)CC and methylates the inner cytosine to produce 5-methylcytosine. Previously isolated relaxed-specificity mutants of the enzyme also methylate, at a lower rate, GG(G/C)CC sites. In this work we tested the capacity of the mutant enzymes to function in vivo as the counterpart of a restriction endonuclease, which can cleave either site. The viability of Escherichia coli cells carrying recombinant plasmids with the mutant methyltransferase genes and expressing the GGNCC-specific Sau96I restriction endonuclease from a compatible plasmid was investigated. The sau96IR gene on the latter plasmid was transcribed from the araBAD promoter, allowing tightly controlled expression of the endonuclease. In the presence of low concentrations of the inducer arabinose, cells synthesizing the N172S or the V173L mutant enzyme displayed increased plating efficiency relative to cells producing the wild-type methyltransferase, indicating enhanced protection of the cell DNA against the Sau96I endonuclease. Nevertheless, this protection was not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the N172S and V173L mutants. Elevated DNA ligase activity was shown to further increase viability of cells producing the V173L variant and Sau96I endonuclease.

scholarly journals Low-level expression of the Type II restriction–modification system confers potent bacteriophage resistance in Escherichia coli

DNA Research ◽  
2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Karolina Wilkowska ◽  
Iwona Mruk ◽  
Beata Furmanek-Blaszk ◽  
Marian Sektas
Keyword(s):  
Dna Methyltransferase ◽  
Host Bacterium ◽  
Type Ii ◽  
Potential Conflict ◽  
Modification System ◽  
Biological Assays ◽  
Restriction Modification System ◽  
Highly Sensitive ◽  
System Maintenance ◽  
Restriction Modification

Abstract Restriction–modification systems (R–M) are one of the antiviral defense tools used by bacteria, and those of the Type II family are composed of a restriction endonuclease (REase) and a DNA methyltransferase (MTase). Most entering DNA molecules are usually cleaved by the REase before they can be methylated by MTase, although the observed level of fragmented DNA may vary significantly. Using a model EcoRI R–M system, we report that the balance between DNA methylation and cleavage may be severely affected by transcriptional signals coming from outside the R–M operon. By modulating the activity of the promoter, we obtained a broad range of restriction phenotypes for the EcoRI R–M system that differed by up to 4 orders of magnitude in our biological assays. Surprisingly, we found that high expression levels of the R–M proteins were associated with reduced restriction of invading bacteriophage DNA. Our results suggested that the regulatory balance of cleavage and methylation was highly sensitive to fluctuations in transcriptional signals both up- and downstream of the R–M operon. Our data provided further insights into Type II R–M system maintenance and the potential conflict within the host bacterium.

scholarly journals Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

2015 ◽  
Vol 44 (2) ◽  
pp. 790-800 ◽  
Author(s):  
Natalia Morozova ◽  
Anton Sabantsev ◽  
Ekaterina Bogdanova ◽  
Yana Fedorova ◽  
Anna Maikova ◽  
...  
Keyword(s):  
Restriction Endonuclease ◽  
Temporal Dynamics ◽  
Modification System ◽  
Restriction Modification System ◽  
Restriction Modification

scholarly journals The Patchy Distribution of Restriction–Modification System Genes and the Conservation of Orphan Methyltransferases in Halobacteria

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 233 ◽  
Author(s):  
Matthew S. Fullmer ◽  
Matthew Ouellette ◽  
Artemis S. Louyakis ◽  
R. Thane Papke ◽  
Johann Peter Gogarten
Keyword(s):  
Dna Methylation ◽  
Gene Transfer ◽  
Dna Methyltransferase ◽  
Dna Uptake ◽  
Modification System ◽  
Selfish Genetic Element ◽  
Restriction Modification System ◽  
Repair Protein ◽  
Restriction Modification

Restriction–modification (RM) systems in bacteria are implicated in multiple biological roles ranging from defense against parasitic genetic elements, to selfish addiction cassettes, and barriers to gene transfer and lineage homogenization. In bacteria, DNA-methylation without cognate restriction also plays important roles in DNA replication, mismatch repair, protein expression, and in biasing DNA uptake. Little is known about archaeal RM systems and DNA methylation. To elucidate further understanding for the role of RM systems and DNA methylation in Archaea, we undertook a survey of the presence of RM system genes and related genes, including orphan DNA methylases, in the halophilic archaeal class Halobacteria. Our results reveal that some orphan DNA methyltransferase genes were highly conserved among lineages indicating an important functional constraint, whereas RM systems demonstrated patchy patterns of presence and absence. This irregular distribution is due to frequent horizontal gene transfer and gene loss, a finding suggesting that the evolution and life cycle of RM systems may be best described as that of a selfish genetic element. A putative target motif (CTAG) of one of the orphan methylases was underrepresented in all of the analyzed genomes, whereas another motif (GATC) was overrepresented in most of the haloarchaeal genomes, particularly in those that encoded the cognate orphan methylase.

scholarly journals Genome sequence of Pseudomonas aeruginosa PAO1161, a PAO1 derivative with the ICEPae1161 integrative and conjugative element

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Adam Kawalek ◽  
Karolina Kotecka ◽  
Magdalena Modrzejewska ◽  
Jan Gawor ◽  
Grazyna Jagura-Burdzy ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genome Sequence ◽  
Dna Methyltransferase ◽  
Laboratory Strain ◽  
Mercury Resistance ◽  
Nucleotide Polymorphisms ◽  
Modification System ◽  
Restriction Modification System ◽  
Integrative Conjugative Element ◽  
Restriction Modification

Abstract Background Pseudomonas aeruginosa is a cause of nosocomial infections, especially in patients with cystic fibrosis and burn wounds. PAO1 strain and its derivatives are widely used to study the biology of this bacterium, however recent studies demonstrated differences in the genomes and phenotypes of derivatives from different laboratories. Results Here we report the genome sequence of P. aeruginosa PAO1161 laboratory strain, a leu-, RifR, restriction-modification defective PAO1 derivative, described as the host of IncP-8 plasmid FP2, conferring the resistance to mercury. Comparison of PAO1161 genome with PAO1-UW sequence revealed lack of an inversion of a large genome segment between rRNA operons and 100 nucleotide polymorphisms, short insertions and deletions. These included a change in leuA, resulting in E108K substitution, which caused leucine auxotrophy and a mutation in rpoB, likely responsible for the rifampicin resistance. Nonsense mutations were detected in PA2735 and PA1939 encoding a DNA methyltransferase and a putative OLD family endonuclease, respectively. Analysis of revertants in these two genes showed that PA2735 is a component of a restriction-modification system, independent of PA1939. Moreover, a 12 kb RPG42 prophage and a novel 108 kb PAPI-1 like integrative conjugative element (ICE) encompassing a mercury resistance operon were identified. The ICEPae1161 was transferred to Pseudomonas putida cells, where it integrated in the genome and conferred the mercury resistance. Conclusions The high-quality P. aeruginosa PAO1161 genome sequence provides a reference for further research including e.g. investigation of horizontal gene transfer or comparative genomics. The strain was found to carry ICEPae1161, a functional PAPI-1 family integrative conjugative element, containing loci conferring mercury resistance, in the past attributed to the FP2 plasmid of IncP-8 incompatibility group. This indicates that the only known member of IncP-8 is in fact an ICE.

Cloning and expression of the NaeI restriction endonuclease-encoding gene and sequence analysis of the NaeI restriction-modification system

Gene ◽  
1995 ◽  
Vol 155 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Christopher H. Taron ◽  
Elizabeth M. Van Cott ◽  
Geoffrey G. Wilson ◽  
Laurie S. Moran ◽  
Barton E. Slatko ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Restriction Endonuclease ◽  
Cloning And Expression ◽  
Modification System ◽  
Restriction Modification System ◽  
Encoding Gene ◽  
Restriction Modification

scholarly journals The Patchy Distribution of Restriction-Modification System Genes and the Conservation of Orphan Methyltransferases in Halobacteria

2019 ◽  
Author(s):  
Matthew S. Fullmer ◽  
Matthew Ouellette ◽  
Artemis S. Louyakis ◽  
R. Thane Papke ◽  
J. Peter Gogarten
Keyword(s):  
Dna Methylation ◽  
Gene Transfer ◽  
Dna Methyltransferase ◽  
Dna Uptake ◽  
Modification System ◽  
Selfish Genetic Element ◽  
Restriction Modification System ◽  
Repair Protein ◽  
Restriction Modification

AbstractRestriction-modification (RM) systems in Bacteria are implicated in multiple biological roles ranging from defense against parasitic genetic elements, to selfish addiction cassettes, and barriers to gene transfer and lineage homogenization. In Bacteria, DNA-methylation without cognate restriction also plays important roles in DNA replication, mismatch repair, protein expression, and in in biasing DNA uptake. Little is known about archaeal RM systems and DNA methylation. To elucidate further understanding for the role of RM systems and DNA methylation in Archaea, we undertook a survey of the presence of RM system genes and related genes, including orphan DNA methylases, in the halophilic archaeal class Halobacteria. Our results reveal that some orphan DNA methyltransferase genes were highly conserved among lineages indicating an important functional constraint, whereas RM systems demonstrated patchy patterns of presence and absence. This irregular distribution is due to frequent horizontal gene transfer and gene loss, a finding suggesting that the evolution and life cycle of RM systems may be best described as that of a selfish genetic element. A putative target motif (CTAG) of one of the orphan methylases was underrepresented in all of the analyzed genomes, whereas another motif (GATC) was overrepresented in most of the haloarchaeal genomes, particularly in those that encoded the cognate orphan methylase.

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